Project 1. Non-homologous end joining (NHEJ) represents the most highly utilized pathway for the repair of DNA double strand breaks (DSBs), which are among the most serious types of DNA damage that the cell must deal with. The NHEJ repair complex forms around the Ku70/Ku80 heterodimer. The N-terminal von Willebrand domain of Ku80 supports interactions with a Ku binding motif (KBM) that has been identified in at least three other DNA repair proteins: the non-homologous end joining (NHEJ) scaffold APLF, the modulator of retrovirus infection, MRI, and the Werner syndrome protein (WRN). A second, more recently identified Ku binding motif present in XLF and several other proteins (the KBMX motif) has also been reported to interact with this domain. In order to understand the structural basis for these interactions, we have studied the isolated Ku80 von Willebrand antigen domain (vWA) from Xenopus laevis. This construct has a sequence that is 60% identical with the human domain, is readily expressed and has been used to investigate these interactions. Structural characterization of the complexes formed with the KBM motifs in APLF, MRI, and WRN identify a conserved binding site that is consistent with previously-reported mutational studies. In contrast with the KBM binding site, structural studies indicate that the KBMX site is occluded by a distorted alpha-helix. Florescence polarization and Fluorine-19 NMR studies of a fluorinated XLF C-terminal peptide failed to indicate any interaction with the frog vWA even at a 1 millimolar concentration. It was hypothesized that availability of this binding site is conditional, i.e., dependent on specific experimental conditions or other repair factors to make the site available for binding. Modulating the fraction of KBMX-accessible binding site mutationally demonstrated that the more open site is capable of binding the XLF KBMX motif peptide. Two of the Ku-binding repair proteins, MRI and WRN, contain both KBM and KBMX motifs and are therefore capable of forming loops with the same Ku80 subunit. It is suggested that the conditional nature of KBMX binding limits non-productive loops, resulting in activation-dependent site availability that may allow either or both of these molecules to bridge two separate Ku-DNA complexes. Project 2. Aprataxin and PNKP-like factor (APLF) is a DNA repair factor containing a forkhead-associated (FHA) domain that supports binding to the phosphorylated FHA domain binding motifs (FBMs) in XRCC1 and XRCC4. We have characterized the interaction of the APLF FHA domain with phosphorylated XRCC1 peptides using crystallographic, NMR, and fluorescence polarization studies. The FHAFBM interactions exhibit significant pH dependence in the physiological range as a consequence of the atypically high pK values of the phosphoserine and phosphothreonine residues and the preference for a dianionic charge state of FHA-bound pThr. These high pK values are characteristic of the polyanionic peptides typically produced by CK2 phosphorylation. Binding affinity is greatly enhanced by residues flanking the crystallographically-defined recognition motif, apparently as a consequence of non-specific electrostatic interactions, supporting the role of XRCC1 in nuclear cotransport of APLF. The FHA domain-dependent interaction of XRCC1 with APLF joins repair scaffolds that support single-strand break repair and non-homologous end joining (NHEJ), and thus apparently competing with the objectives of each scaffold-supported repair process. It was suggested that this overlapping interaction results in competitiverepair pathways that compete to optimize the fidelity of the resulting repair, as has been discussed by Iliakis and coworkers. Project 3. DNA ligation is a central process in biology that finalizes genome maintenance metabolic processes including DNA replication, recombination, and DNA repair. Eukaryotic DNA ligases catalyze ligation via a three-step, ATP-dependent reaction. First, the DNA ligase active site lysine is adenylated. Second, the adenylate is transferred to a DNA 5' phosphate to facilitate the nick-sealing step. Third, nucleophilic attack of a 3'-OH on the activated 5'-adenylate facilitates phosphodiester bond formation, and sealing of the DNA break. Environmental and metabolic sources of DNA damage can result in abortive ligation, due to the failure to complete step 3, with the resulting generation of 5'-adenylated (5'-AMP) DNA strand breaks. The aprataxin (APTX) RNA-DNA deadenylase protects genome integrity and corrects abortive DNA ligation arising during ribonucleotide excision repair and base excision DNA repair, and APTX human mutations cause the neurodegenerative disorder ataxia with oculomotor ataxia 1 (AOA1). How APTX senses cognate DNA nicks and is inactivated in AOA1 remains incompletely defined. We have determined structures of APTX engaging nicked RNA-DNA substrates that provide direct evidence for a wedge-pivot-cut strategy for 5'-AMP resolution shared with the alternate 5'-AMP processing enzymes DNA polymerase beta and flap endonuclease 1 (FEN1). These studies reveal a DNA-induced fit mechanism regulating APTX active site loop conformations and assembly of a catalytically competent active center. We also have defined a complex hierarchy for the differential impacts of the AOA1 mutational spectrum on APTX structure and activity. Sixteen AOA1 variants impact APTX protein stability, one mutation directly alters deadenylation reaction chemistry, and unexpectedly, a dominant AOA1 variant allosterically modulates APTX active site conformations. As noted above, aprataxin contains a histidine triad (HIT) nucleotide hydrolase whose function can be perturbed by mutations that have been associated with patients afflicted with AOA1. We recently initiated a further, more detailed NMR investigation of the behavior of the catalytic histidines of the enzyme aimed at better understanding the molecular basis for functional impairment by many of these reported mutations. As is often the case, the basis for functional impairment by some of these mutations is straightforward, while the mechanism by which other mutations interfere with catalytic function is much less obvious. It is anticipated that these additional studies will provide a more detailed and sensitive basis for understanding the effects of these additional mutations. Project 4. Folate-dependent enzymes play a central role in single carbon metabolism and in nucleotide biosynthesis; its perturbation can be mutagenic and ultimately lethal. More generally, dysregulation of folate metabolism appears to be broadly linked to deficiencies in genome methylation, stability and repair. Alternatively, anti-folate drugs such as methotrexate are widely used in the treatment of neoplastic disease, but low doses have found increasing application in the treatment of rheumatoid diseases and other illnesses that are associated with chronic inflammation. Our previous studies of folate metabolism indicated that the NSAID fenbufen was able to interact with the enzyme dihydrofolate reductase (DHFR) by binding to the region of the folate binding site that interacts with the p-aminobenzoyl-L-glutamate (pABG). We are currently performing a more general assessment of the interaction of other NSAIDs with this subsite, their ability to bind to this site, and their inhibition of DHFR activity. These studies have quantified the DHFR inhibitory strength of various NSAIDs, and demonstrated that some possess even greater inhibitory potency than fenbufen. The potential anti-folate activity of NSAIDs suggests the possibility of additional applications in the treatment of chronic inflammatory diseases without some of the toxicity of more potent antifolate compounds such as methotrexate.